CHAPTER 1
Antibiotic Resistance Profiling
Stephen J. Hammonds
1. Introduction
Antibiotic resistance is not an absolute property. Even within a single
species, strains can exhibit different degrees of antibiotic resistance.
When a bacterium is tested for resistance, it should be compared, quan-
titatively with a standard. This standard may be the resistance of a wild-type
strain, a typical strain, or a “type” strain, appropriate to the test strain.
Most commonly in these studies, large numbers of test isolates are
screened for their resistance on one or more antibiotics. The test strains
may be mutant or recombinant derivatives of a known parental strain, or
they may be fresh isolates from an independent source. The degree of
resistance is quantified by incorporating known concentrations of an
antibiotic into solid growth medium. The bacterial test strains are then
inoculated onto the medium. After a period of growth, the plates are
examined for growth and the lowest antibiotic concentration that inhibits
the growth of each bacterium is determined. This measure is the minimum
inhibitory concentration (MIC). This is then compared with the observed
MIC of a standard strain or with a reference threshold value (I).
This is an accurate quantitative technique that uses well-established
culture and test methods. In addition, the expected MIC values for many
different species of bacteria are readily available in the literature (2). The
method described here is suitable for bacterial species commonly used in
genetic studies and genetic engineering, such as
Escherichia coli.
The
technique is readily adaptable to other species by adjusting the media
and culture conditions appropriately. However, the effect of such changes
on the activity of the antibiotic should be considered carefully (see Note 1).
From: Methods In Molecular Biology, Vol 46: Dagnostlc Bacteriology Protocols

Edited by J Howard and D M. Whltcombe Humana Press Inc , Totowa, NJ
1
2
Hammonds
The technique described is based on the standard method used in the
United Kingdom for determination of the sensitivity of the pathogenic
bacteria to antibiotics. It forms the basis of the well-known “breakpoint”
method for sensitivity testing, widely employed in diagnostic bacteriol-
ogy laboratories. The antibiotics that are most appropriate for testing
against the various species of pathogenic bacteria may be found in
Lambert and O’Grady (2).
The technique may also be used to determine the sensitivity of bacte-
ria to a range of antibiotics to obtain a pattern of antibiotic sensitivities
known as an antibiogram. These antibiograms may be used in bacterial
taxonomy.
2. Materials
1. Phosphate-buffered saline (PBS): 20 mi!4 sodium phosphate, pH 7.4,
0.85% (w/v) NaCl. Sterihze by autoclaving at 121°C for 15 min. Store at
4°C indefinitely, but warm to room temperature before use as a culture dil-
uent, because the viabthty of some strains can be affected by cold diluent.
2. Phosphate buffer: 20 mM sodium phosphate, pH 7.0. Sterilize by auto-
claving at 121°C for 15 min and store indefimtely at 4°C.
3. Antibiotics: 1 mg/mL in sterile 20 mM phosphate, pH 7.0. Sterilize by
filtration through a 0.22~pm filter and store for up to 24 h at 4°C. Any
antibiotics may be used, but solubihty in water 1s crucial. This may require
the use of antibiotrc salts (see Note 2). Antibiotic preparations supplied for
clinical use are often unsuitable, because they are sometimes inactive in
vitro. Furthermore, they often contam additional, nonantibiotic substances
that could interfere with the assay. Details of the stability and storage
conditions can be obtained from the suppliers. In general, though, the

undissolved powders should be kept desiccated, m the dark and at low
temperature.
4. Liquid medium: Any rich medmm suitable for the organism under study.
For many strains, tryptone soya broth (TSB), also known as tryptrcase soy
broth, is suitable.
5. Test agar: Mueller-Hmton agar and dragnostic sensitivity agar have been
developed specifically for antibiotic testing purposes and are commercially
available. Dtssolve the agar mix according to the manufacturer’s mstruc-
tions and heat to 100°C for 5-10 min to dissolve the solids. Adjust the pH
to exactly 7.4 and autoclave at 121°C for 15 mm. Allow the agar to cool
and keep it, molten, at 45°C until required. Prepare the molten agar on the
day of use. Other media can be used but be sure that the components of
such media have no interfering effects on antibiotic activity (see Note 1).
Antibiotic Resistance Profiling
3
3. Method
1. Inoculate 10 mL of TSB with the test strains. Always mclude at least one
control or reference strain (often the parental strain from which the test
isolates were derived).
2. Grow the cultures at then optimal temperature to early stationary phase
(usually 18-24 h). Vary the mcubation temperature and time as appropri-
ate to the strain. The anticipated cell density is 10’ viable cells/ml.
3. Dilute the cultures 1 in 100 in PBS (see Note 3).
4. Immediately before use, prepare a series of five lo-fold dilutions of the
antibiotic in 20 mM phosphate. Always use a fresh tip for each dilution to
avoid carryover of concentrated antibiotic solution to dilute.
5. Dispense 2 mL of each dilution mto premarked Petri dishes. Also, pipet
2 mL of neat antibiotic solution and 2 mL of phosphate buffer only into
two more dishes.
6. Dispense 18 mL of molten agar mto each dish and thoroughly mix in the

antibiotic by swirling the plate on the bench.
7. Allow the agar to set at room temperature and chill the plates at 4°C to
harden the media.
8. Dry the plates m a plate-drying oven at 55°C for 20 min.
9. Spot each of the diluted cultures onto each of the antibiotic dilution plates,
using sterile loops or applicator sticks (see Notes 1 and 3). Be sure to mark
the onentatton of the plates to allow later identification of the spots. It may
be helpful to use a template where many spots are to be tested.
10. Invert the plates and incubate them under the appropriate conditions
(typically, 37°C for 18-24 h).
11. Examine the surface of the plates and determine the lowest concentration
of antibiotic that inhibits growth of each strain (MIC). Ensure that the
control and/or reference strains have produced the anticipated results.
12. Determine the MIC more accurately by using a twofold dilution series of
antibiotrc, starting with the dilution above the MIC.
4. Notes
1. The activity of many antibiotics 1s significantly affected by the physical
and chemical conditions in the bacterial culture medium (3):
a. Acid conditions, which arise by fermentation of carbohydrate m the
medium, may reduce the activity of lincomycin, erythromycin,
cephaloridine, and the aminoglycosides;
b. Alkaline conditions reduce the activity of methicillin, cloxacillin,
fucidin, and the tetracyclmes;
c. Anaerobic conditions reduce the activity of ammoglycosides;
Hammonds
d. Aerobic conditions reduce the activity of metronidazole;
e. Divalent cations such as magnesium and calcmm reduce the activity of
aminoglycosides and tetracyclines against Pseudomonas
aeruginosa;
and

f. Pyrtmidines and related compounds reduce the acttvtty of sulfonamtdes
and trimethoprtm.
2. Some antibrotics are insoluble in water in the form of the base. However, tt
is often the case that soluble salts exist for these antibiotics (e.g., erythro-
mycin lactobionate, trimethoprim lactate, and polymyxin sulfate). It is
sometimes also possible to dissolve the base m a minimal volume of 0. 1M
NaOH (e.g., nitrofurantoin, nalidixtc acid, and the sulfonamides), or O.lM
HCl (e.g., rifampicm), or ethanol (e.g., chloramphenicol, erythromycin)
before dilution to the required volume with 20 mM phosphate buffer.
Details of the solubibty and other properties of antibiotics may be found m
The Merck Index (4).
3. The concentration of the moculum is less important in the agar incorpora-
tion technique than in other methods. However, too high a concentration
of bacteria does cause a sigmftcant reduction in the activity of
sulfonamides. Consequently, the correct dilution of bactertal suspension
must be used.
References
1 Phillips, I. (1991) A guide to sensitivity testing. Report of the working party on
antibiotic sensitivity testing of the British Society for antimicrobial chemotherapy
J. Antimicrob Chemother 27(Suppl. D), l-50.
2 Lambert, H. P. and O’Grady, F (1992) Antibiotic and Chemotherapy, 6th ed
Churchill Livingstone, Edinburgh, UK.
3. Houang, E T., Hince, C , and Howard, A. J. (1983) The effect of composition of
culture media on MIC values of antibiotics, in Antibiotics: Assessment of
Antimicrobial Activity and Resistance (Russell, A. D. and Quesnel, L B , eds ),
Academic, London, pp. 3 l-48.
4 Budavari, S. (1989) The Merck Index. Merck, Rahway, NJ.
CHAPTER 2
Bacteriocin Typing
l)rone L. Pitt and Michael A. Gaston

1. Introduction
Bacteriocins are proteins produced by bacteria which are lethal for other
members of the same species and, occasionally, for other species. In gen-
eral, bacteriocins are active in very low concentrations against specific
strains. This property has been widely utilized for the identification of
types of strains within several bacterial species and most methods are
based on the inability of a strain (indicator) to grow on an agar surface that
has previously supported the growth of the bacteriocin producer strain.
Bacteriocin typing is a simple, but powerful, technique to distinguish
between strains of bacterial species but is often overlooked in favor of
more technically sophisticated methods. At its simplest level no stored
reagents or even reference strains are required, as a collection of isolates
can be tested against each other for bacteriocin production/sensitivity.
The information gained in this way is likely to be less valuable than that
obtained with an established reference typing method but may be suffi-
cient for the identification of unique strains in an outbreak situation.
1.1. Nomenclature
The naming of bacteriocins has been problematic. Some workers prefer
to use the term bacteriocin of, for example,
Klebsidu
or
Pseudomonas,
whereas others refer to klebocin or pyocin, respectively. This trivial form
of nomenclature is widely used but the discovery of new bacteriocins may
lead to taxonomic confusion.
From’ Methods m Molecular B/ology, Vol. 46. D!agnost/c Bacteriology Protocols
E&ted by J Howard and D. M Whltcombe Humana Press Inc , Totowa, NJ
5
Pitt and Gaston
1.2. Characterization

Bacteriocins are a large heterogeneous group of antibiotic substances
that differ greatly in their molecular size and their resistance to heat,
chloroform, and proteolytic enzymes. Three basic groups have been
defined:
1. Cohcin-like agents ranging 111 molecular size from 3 x lo6 to 1 x lo8 Daltons;
2. Bacteriophage-like particulate bacteriocins that resemble phage tails, e.g.,
R (retractile) and F (flexuous) pyocins of Pseudomonas aeruginosa; and
3. Low molecular weight bactenocms, also called mlcrocins, which are bac-
tericidal peptldes (I-3)
1.3. Activity Spectra
The bacteriocins of Gram-negative bacteria often have a narrow spec-
trum of activity. In contrast, the bacteriocins produced by Gram-positive
bacteria have a broad host range and are sometimes active on taxonomi-
tally unrelated genera (4).
1.4. Mode
of
Action
The modes of action of some bacteriocins have been established. For
example, colicins cause specific inhibition of protein synthesis or induce
a single endonucleolytic break in RNA, whereas others inhibit DNA syn-
thesis (5). Some bacteriocins inhibit macromolecular synthesis by
uncoupling electron transport systems or cause disruption of the perme-
ability barrier of the cell wall by enzymatic action, as is the case with the
megacins of Bacillus megaterium. Specific receptors are required for
the adsorption of bacteriocins and resistant strains often lack, or have
occluded, surface receptors. Receptors that have been identified include
glycoproteins, outer membrane proteins, and lipopolysaccharides.
1.5. Resistance and Tolerance
A bacteriocin is inactive against strains possessing the same
bacteriocinogenic factor. These factors may be plasmid borne or chro-

mosomal. Strains may produce more than one type of bacteriocin, but
the immunity of producer strains to bacteriocins is often extremely
specific and they may be sensitive to a closely related bacteriocin. Some
bacteria produce a specific inhibitor protein that complexes with the bac-
teriocin and protects the producer strain from its action. Alternatively,
immunity may be associated with the inability of the producing bacterium
to adsorb homologous bacteriocin (6). A distinction should be made
Bacteriocin Typing 7
between resistant and tolerant strains; the former do not adsorb bacterio-
tin, as do tolerant strains, but the lytic action is blocked subsequently.
1.6. Practical Considerations
The inhibition of strains by bacteriocins can be confused with the
action of bacteriophages and vice versa. In some strains this can be fur-
ther complicated by the simultaneous production of both bacteriocin and
bacteriophage. A simple way to distinguish between the two groups is to
prepare a logarithmic dilution series of a suspension containing the
inhibitor and spot a sample of each dilution onto the indicator strain.
Inhibition owing to bacteriocins will generally only be visible to a
dilution of lo3 or lo4 and will “fade out,” whereas bacteriophages will
usually be active to at least a dilution of lo7 and will show individual
plaques at high dilutions.
1.7. Typing Schemes
Bacteriocin typing schemes can be classified as active or passive
methods. In the first, the bacteriocins produced by the test (field) strains
are detected by their activity on a panel of standard indicator strains,
whereas in the second method, bacteriocins from a set of producer strains
are tested for activity on the field strains. The discrimination achieved by
either method should be determined by experiment as well as the sta-
bility (reproducibility) of the patterns of inhibition. Schemes with
high reproducibility will allow patterns to be designated as types, but

this is impractical for those schemes that exhibit excessive variation on
repeated testing.
Three different methodological approaches have been used for routine
typing of isolates. The original method of “scrape and streak” (7),
described in Section 3.3., is not commonly used today because it is too
labor-intensive; it takes 3 d to complete and reactions can be equivocal.
Essentially, the bacteriocin-producing strain is grown in a streak across
the diameter of an agar plate. Following incubation, this growth is
removed and the indicator strain is inoculated at a right angle over the
original streak. Bacteriocin activity is evidenced by inhibition of the
indicator strain.
In the lysate method (B), described in Section 3.2., bacteriocins of a
producer strain are induced by mitomycin C and cell free lysates are
spotted onto lawns of the indicator strain. This method is not widely used,
perhaps because of the need to prepare lysates that not only require stan-
8
Pitt and Gaston
dardization of bacteriocin concentration but also deteriorate on storage.
The influence of bacteriophages that are also induced by mitomycin C
can be reduced by UV irradiation of the lysate, but this is a time-consum-
ing and variable procedure.
The principle of the overlay method (9) (see Section 3.1.) is that spots
of a dense suspension of bacteria containing preformed bacteriocin are
allowed to grow for a short period in which more bacteriocin is formed.
The thin agar layer serves to concentrate the bacteriocins around and
under the spot of growth. Bacteriocin activity is visualized by inhibition
of the growth of the indicator strain contained in the overlay; diffusion is
enhanced by the use of soft agar. This method also allows the differen-
tiation of low molecular weight diffusible bacteriocins from the particu-
late high molecular weight structures by their inhibition zone diameters.

The major advantages of this procedure over others is the rapidity and
the clarity of reactions that can be obtained.
1.8. Prior Considerations
The choice of methodology for bacteriocin typing is greatly influ-
enced by the growth characteristics of the species to be examined and
the equipment available. We recommend that the following factors
are determined by experiment in order to optimize conditions for a par-
ticular species.
1.8.1. Media
Some media constituents are inhibitory to bacteriocin production.
For example, bile acids are antagonistic to Proteus mirabilis bacterio-
tins (10) and, therefore, McConkey agar is unsuitable for bacteriocin
typing of this species. Other compounds that have been reported to affect
bacteriocin synthesis include casein, amino acids, manganese, and sug-
ars, particularly glucose (4). The addition of enzyme inhibitors such as
iodoacetic acid has been advocated to improve the clarity of inhibition
reactions (11).
1.8.2. Temperature
Bacteriocins are often produced in higher yield if the strain is
incubated at a temperature slightly below the growth optimum. This
serves two purposes; first, it reduces the amount of cell growth and, sec-
ond, it delays the production of bacteriocin, inactivating substances such
as proteases.
Bacteriocin Typing 9
1.8.3. Induction
Most bacteriocins may be recovered in higher yield if an inducing
agent is used. Mitomycin C is the agent most commonly used. This
compound damages DNA and, as a result, a complex cellular mechanism,
the SOS regulatory system, is activated. DNA repair occurs in the absence
of template instruction resulting in the creation of many nonsense or

deletion mutations. Lysis of these mutants occurs at a rapid rate with a
release
of
cellular contents that include bacteriocins and lysogenic phages.
Most workers have used mitomycin C to induce bacteriocin production
in liquid media but care should be taken to rule out the contribution of
phage in the lysate to the inhibition zone formed on agar lawns of suscep-
tible bacteria. Anaerobiosis can also induce bacteriocin synthesis and nitrate
can be incorporated into a growth medium to encourage anaerobic growth
of Pseudomonas (nitrate acts as an alternative electron acceptor when
cleaved by nitrate reductase to produce molecular oxygen). Bacteriocin-
inactivating substances may also be suppressed by anaerobiosis and recep-
tors may not be synthesized in the absence of oxygen.
2. Materials
1. Nutrrent broth and solid media appropriate to the growth requirements of
the species (see Note 1). In the agar overlay method, add 0.5 g of bacterro-
logrcal grade agar to 100 rnL of 1% (w/v) peptone, boil for 15 mm to
dissolve the agar, and sterilize at 121°C for 15 min. Drspense into sterile
bijoux bottles in 2.5-mL volumes.
2. Mitomycin C: 100 ug/mL in sterile water, store m lOO-200~yL aliquots in
foil-wrapped bottles at -70°C. Discard the stock after 6 mo and do not
refreeze. This 1s a hazardous agent and appropriate safety precautions
should be taken.
3. Saline: 0.2M in distilled water. Sterilize by autoclavmg and store at room
temperature.
4. Chloroform (see Note 2).
5. Multipomt inoculator (see Note 3).
6. Membrane filters: 0.45 urn pore size, sterilizing grade.
3. Methods
3.1. Agar-Overlay Method (Fig. 1)

1. Pipet 10 mL of molten agar into g-cm diameter Petri dishes. Allow the
agar to set and dry the surface at 37°C for 15 min. Store plates m a sealed
plastic bag at 4°C overnight.
10
Pitt and Gaston
Bacteriocin productlon
Bacteriocin detection
Fig. 1. Bacteriocm typmg by the method of Fyfe et al. (9).
2. At the same time, grow the bacteriocin-producing strams overnight on
nutrient agar plates.
3. Disperse 5-10 colonies with a cotton swab m 2 mL of sterile distilled water
to give a smooth suspension of approximately lOlo cfu/mL.
4. Dispense the suspensions into wells of a sterile reservoir block and apply
spots of 0.3-0.5 pL with a multipomt moculator onto the surface of the
lo-mL agar plates.
5. Incubate plates at a suitable temperature (see Note 4).
6. Remove plates from incubator and, m a fume hood, invert the agar surface
for 15 min over filter paper pads (30 x 30 mm) that have been impregnated
with 0.5 mL of chloroform (see Note 5).
7. Place the plates, open, in a 37OC incubator for 30 min to dry the surface
and dispel all chloroform vapor.
8. Add 0.05 mL of a log-phase broth culture of an indicator strain to 2.5 mL
of molten overlay agar at 50°C.
9. Mtx gently and pour the total volume rapidly over the entire surface of
the plate.
10. Incubate the plate overnight at the optimal temperature for growth and
record zones of inhibition.
3.2. Lysate Method
1, Grow the producer strains m broth overnight.
Bacteriocin Typing

11
2. Dilute 200 p,L of thts broth culture into 2 mL of fresh broth and incubate
the tubes m a shaking water bath at an appropriate temperature until log-
phase growth is achieved (see Note 6).
3. Add mitomycin C to a final concentration of 1 .O l.tg/niL and continue the
incubation, with shaking, for 6 h. There is usually an inmal increase in
turbidity followed by a decline.
4. Add 0.5 mL of chloroform and shake the tubes vigorously.
5. Leave the tubes at room temperature for 15 min to allow the phases to
separate.
6. Centrifuge the tubes at 5OOOg for 20 min and recover the aqueous phase.
7. Filter the supernatant through a sterilizing grade membrane filter (0.45 pm)
and store at 4°C.
8. Make a dilution series of filtrate in l-mL volumes of saline. Make lo-, 50-,
loo-, 200-, 400-, 800-, lOOO-, and lO,OOO-fold dilutions m I-mL volumes
of saline.
9. Pour a lawn of 2.5 mL of overlay agar (5O’C) containing 0.05 mL of a log-
phase broth culture of the indicator strain over the surface of a dried
(25 rnL) nutrient agar plate.
10. Allow the soft agar to set and dry the surface at 37°C for 10 min.
11, With a micropipet, dehver 20 l.tL of each of the dilutions of lysate onto the
agar surface.
12. Leave the plate open to dry for 10 mm.
13. Incubate the test plate overnight at an appropriate temperature.
14. Calculate the bacteriocm titer: For these purposes, the titer is defined as
the highest dilution of lysate that inhibits the growth of the indicator strain,
and is expressed as the number of bacteriocin U/20 FL. Multiply this figure
by 50 to give the number of U/r& For the typing of clinical isolates, dilute
each lysate to give a standard predetermined bacteriocin concentration
(see Note 7).

3.3. Scrape and Streak Method (Fig. 2)
1. Grow the producer strain to log-phase in broth medium.
2. Inoculate, with a sterile cotton swab, a strip (approx 1 cm wide), across the
diameter of an agar plate.
3. Incubate the plate overnight at a suitable temperature (see Note 4).
4. Scrape off the growth with a microscope slide, taking care not to damage
the surface of the agar.
5. Expose the agar surface to chloroform vapor for 15 mm (see Section 3.1.,
step 6).
6. Leave the plate open, m air, for 15 min to drive off all chloroform.
7. Prepare log-phase broth cultures of the indicator strains.
Pitt pnd Gaston

Fig. 2. Bacteriocin typing by the method of Abbott and Shannon (7).
8. Dispense 50-p,L volumes into a multiwell plastic block or microtitration tray.
9. Streak the indicator strains at right angles across the area of agar originally
covered by the producer strain (see Note 8).
10. Incubate at the optimal growth temperature and record the inhibition reac-
tions as posmve or negative (see Note 9).
3.4. Interpretation of Bacteriocin Typing
Bacteriocin typing is most often performed with reference to estab-
lished type schemes and therefore the protocol for the identification of
types will depend on the species under investigation. For example, for
P.
aeruginosa,
the activity of pyocins produced by field strains on a panel
of eight indicator type strains and five subtype strains is recorded as + or -,
and the pattern of inhibition is matched with a standard table that defines
105 types (12). Patterns of strains not contained in the table may be given
arbitrary type designations and these can be used as part of a local data-

base to classify subsequent isolates. In theory, the number of types may
only be limited by the number of possible combinations of 13 indicators.
This approach to bacteriocin typing is restricted to systems that have
high reproducibility, as excessive variation in patterns will invariably
place the same isolate in different types on repeated tests.
For some species, however, the type is determined by the profile of the
bacteriocins produced by the field strain against a panel of indicator
Bacteriocin Typing 13
strains as well as the sensitivity of the field strain to bacteriocins
produced by a set of standard strains. This form of production/sensitivity
(P/S) typing is used for Proteus spp (13) and P. cepacia (14). Field strains
are allocated a P/S type according to a reference table.
The use of short numerical codes or mnemonics has been advocated
where a large number of reactions are compared (15). For example, a
strain with a type pattern with nine indicators of “+-+ + + ” would
be coded 375 (+-+ = 3, + = 7, and + = 5) as each combination of
reactions within each triplet is identified by a number. Alternatively, each
indicator in the triplet may be numbered 4,2,1, respectively, and so +-+
would = 5, + = 1, and + = 4; reactions with all indicators in the
triplet would be coded 7, and so on.
4. Notes
1. The agar for the growth of the bacteria should be formulated to give a clear
medium, especially for the overlay method, and it should support the
confluent growth of the species under test.
2. Plastic Petri dishes can be used for bacteriocin typing if care is taken with
the chloroform treatment stage (see
also
Note 5). Previously, most workers
used glass plates but these are fragile, and require washing and sterilizing.
3. The multipoint inoculator should deliver 19 or 36 discrete spots. If this

equipment is not available the spots can be applied with a micropipet or
with a l-mm diameter loop. Manual application of cultures is best achieved
with the aid of a paper backing template cut to the diameter of the Petri
dish and marked with equally spaced dots.
4. For fast-growing bacteria, incubate for 5-6 h at 5-7°C below the optimum
growth temperature. Growth should be minimal after incubation and just
visible to the naked eye.
5. Ensure that the chloroform does not come into contact with the plastic
plate. Clouding of the plastic, with subsequent melting, will occur owing
to chloroform vapor. This is reduced if the volume of chloroform and time
of exposure is kept to a minimum.
6. The turbidity may be monitored spectrophotometrically, but for most fast-
growing aerobic species, 1.5-3 h is optimal.
7. Some bacteriocins may give consistently weak inhibition zones even at
high concentrations. Double-agar layered plates are not necessary for all
species and a conventional seeded lawn is often suitable.
8. The streak of the indicator strain may be made to the midpoint of the origi-
nal inoculum of the producer strain or entirely across the agar surface. A
variety of mechanical aids have been developed to facilitate the simulta-
14 Pitt and Gaston
neous cross-streaking of a number of indicator strains. The simplest is a
comb of loops fixed at a right angle in a metal rod with a handle.
9. Zones of inhibition are sometrmes not clear and may be difficult to read.
Reactions can vary from full inhibition through a thinning of growth to the
presence of isolated colonies in the inhibition zone. This may be mini-
mized by diluting the broth culture of the indicator strain 1 m 10 or 1 m
100. Phage activity is occasronally seen m the inhibition zone as patches of
incomplete lysis.
References
1. Konisky, J. (1982) Colicins and other bacterrocms with estabhshed modes of

action, in Annual Revtew of Microbiology (Starr, M P., ed.). Annual Reviews,
Inc., Palo Alto, CA, pp 125-144
2. Bradley, D. E. (1967) Ultrastructure of bacteriophages and bacteriocins. Bacterial
Rev. 31,230-314
3. Baquero, F. and Moreno, F. (1984) The microcms FEMS Microbial. Lett 23,
117-124
4 Tagg, J. R., Dajani, A S., and Wannamaker, L W. (1976) Bacterrocms of Gram-
positive bacteria Bacterial. Rev 40,722-756.
5. KeIl, D. B , Clarke, D. J., and Morris, J. G (1981) Proton coupled information
transfer along the surface of biological membranes and the mode of action of cer-
tain cohcins. FEMS Mtcrobiol Lett. 11, 1-12
6. Levisohn, R., Komsky, J., and Fomura, M. (1968) Interactron of cohcms with bac-
terra1 cells. IV. Immunity breakdown studied with colxins la and lb. J. Bactenol
96,811-821.
7. Abbott, J. D and Shannon R. (1958) A method of typing Shigella sonnei using
colicine production as a marker J. Clin Pathol. 11,71-77.
8. Falkiner, F. R and Keane, C. T. (1977) Epidemiological information from active
and passive pyocine typing of Pseudomonas aeruginosa. J. Med Microbial. 10,
447-459
9. Fyfe, J. A. M , Harris, G., and Govan, J. R. W. (1984) Revised pyocm typing
method for Pseudomonas aeruginosa. J Clin. Mtcrobiol. 20,47-50.
10. George, R. H. (1975) Comparison of different medra for bacterrocin typing of Pro-
teus mirabilis J. Clin Pathol. 28,25-28
11. Darrell, J. H. and Wahba, A H (1964) Pyocme-typing of hospital strains of
Pseudomonas aeruginosa. J. Cltn Pathol. 17,236-242
12. Govan, J. R. W. (1978) Pyocin typing of Pseudomonas aeruginosa, in Methods in
Microbiology, vol. 10 (Bergan, T. and Norris, J., eds.), Academic, London,
pp. 61-91.
13 Senior, B W (1977) Typing of Proteus strains by proticme production and sensr-
tivity. J. Med Mtcrobiol 10,7-17.

14. Govan, J. R. W. and Hams, G (1985) Typing of Pseudomonas cepacta by
bacteriocin susceptibility and production. J. Clin Microbial. 22,490-494.
15. Farmer, J. J. (1972) Epidemiological differentration of Serratia marcescens typ-
ing by bacteriocin production. Appl. Microbtol. 23,218-225
CHAPTER
3
Bacteriophage Typing
@-one L. Pitt and Michael A. Gaston
1. Introduction
Bacteriophages (phages) are viruses that infect bacteria. Susceptibility
to infection by particular phages varies between strains within a species,
and this property can be exploited to construct highly discriminatory
schemes for the type identification of strains in epidemiological studies.
Part of the success of the technique is due to the simple methodology
involved. Production and handling of bacteriophages requires nothing
more than a competent aseptic technique and a basic understanding of
their characteristics and life cycle.
1.1. Bacteriophage Life Cycle
Phages infect bacteria by attachment to specific receptors and injection
of nucleic acid into the cell. In the extracellular state, a phage exists as a
metabolically inert particle or
virion,
which is a protein coat (capsid)
surrounding nucleic acid, DNA or RNA. The nucleic acid may be double-
or single-stranded, in a linear or circular configuration (1,2). The
following two situations may arise on injection of phage DNA into the
host cell:
1. The DNA may become stably integrated into the bacterial chromosome.
This is referred to as
Zysogeny,

and phages capable of this are termed
temperate
phages. In lysogeny, a small proportion of host cells do express
the phage genes and some lysis and hberation of phage progeny occur,
2. The DNA may enter a rephcative cycle, leadrng to the death of the host
and the production of new phage particles. These
Zytic
or
virulent
wild
phages lyse the bacteria at the end of the rephcative cycle and release a
From Methods m Molecular Biology, Vol 46. Dlagnostlc Bacteriology Protocols
Edtted by J Howard and D M Whltcombe Humana Press Inc , Totowa, NJ
15
16 Pitt and Gaston
large number of daughter phage particles that infect neighboring cells. This
process leads to the formation of a plaque or visible inhibition of growth of
the host cell (3).
The host range
of a phage is governed by a number of complex factors.
The appropriate receptors must be accessible to the phage and, following
infection, the phage DNA must evade host restriction
and modification
enzymes, which recognize foreign DNA and prevent its incorporation
into the bacterial genome. Furthermore, bacteria that have previously
been lysogenized by a phage are immune to lysis by related phages.
1.2. Practical Considerations
Phages may be modified or adapted to exhibit different host ranges
from parent phages by propagation in different hosts of the same spe-
cies,

In general, phage typing systems based on adapted phages are
more reproducible and strain specific than those that utilize wild or
temperate phages.
Phage typing schemes can offer very high levels of discrimination
between strains, although reproducibility is a recurring problem. For many
species, a large number of phages with different specificities can be iso-
lated readily from both the environment and lysogenic cultures. This num-
ber may be reduced by study of lytic spectra to form manageable sets for
phage typing.
Phages are easy to handle in a laboratory with only basic equipment and
can, in most cases, be propagated to produce large volumes of high titer
stock that can be stored at 4°C for many years. Lytic reactions are not
usually difficult to read and can be recorded in a manner that allows iso-
lates to be readily compared with each other.
1.3. Development
of
a Phage-Typing Scheme
The development of a phage scheme can be divided into a number
of stages.
1.3.1. Selection of Strains for the Isolation of Phages
The selection of the initial strains to be used for the isolation of phases and
for the panel of test strains is one of the most important steps in the estab-
lishment of a typing set. The larger the number of strains and the more rep-
resentative they are of the overall population, the greater the chance of
selecting a suitable set of typing phages. Schemes either can be devel-
oped as primary classification systems or as secondary typing meth-
Bacteriophage Typing
17
ods, and these strategies require different sets of strains on which to develop
and evaluate the scheme.

1.3.2. Isolation of Candidate Phages
If environmental sources are available, then try as many varied sources
as possible. For example, sewage treatment plants, animal waste mate-
rial, general farm slurry, and water.
1.3.3. Evaluation and Selection of Most Efficient Typing Set
In order to achieve a selection of typing phages that give good
discrimination, candidate phages must be evaluated on a large panel of test
strains. This gives rise to a large amount of data requiring careful analysis.
In fact, the selection of typing phages is the most difficult part of establish-
ing a phage scheme, There is no substitute for subjective analysis of the
data, but there are relatively simple objective methods that can greatly ease
the burden of analysis.
We suggest the following approach to selection.
1. Discard phages with unsatisfactory lytic reactions. Many phages give very
turbid plaques that would be inappropriate in a routine typing set.
2. Discard phages that only lyse a small percentage of strains (<5%).
3. Discard phages that exhibit rapid loss of titer (see Note 1).
4. Perform numerical analysis with the Jaccard coefficient (4). Similarity data
and dendrograms are a quick route to the discarding of phages with very
similar lytlc patterns (5,6).
5. Perform subjective analysis of lytic reactions of the remaining phages. It
may be useful to do a more extended analysis with a provlslonal typing set
before choosing a final set.
1.3.4. Statistical Approaches to Phage Selection
There are a number of computer applications that can aid the selection
of tests for phage typing schemes. Briefly, some of the approaches used by
taxonomists for the interpretation of biochemical tests can be extremely
useful in the analysis of lytic reactions. The operational taxonomic unit,
conventionally a bacterial strain, is replaced by a phage, and the characters,
usually the results of biochemical tests (positive or negative) are replaced

by the results on test strains: A positive result is scored if one of the panel
of test strains is lysed by the phage. In the context of choosing suitable
typing phages, numerical taxonomy is essentially a negative selection pro-
cedure: It is a useful indicator of related phages, most of which can be
excluded from a typing set on the basis of redundancy.
18
Pitt and Gaston
The positive selection of phages is best carried out sequentially, start-
ing with the most discriminatory phage. This is the phage with the highest
separation figure. The separation figure is the number of positive reac-
tions multiplied by the number of negative reactions. Thus, given a set of
100 strains, the maximum separation figure is 50
x
50 = 2500, so the
most useful phage is the one that reacts with approximately 50% of the
strains. This parameter is easy to calculate and is a useful datum on indi-
vidual phages.
A more powerful approach is to use a computer program that will
choose phages sequentially, starting with the most discriminatory iso-
late, and adding phages so as to maximize the number of strain pairs
distinguishable by the partial set (7).
1.3.5. Storage of Strains and Phages
Once the scheme has been finalized, all the propagating strains and the
phages must be freeze-dried. This not only provides a useful safety net
should a strain or phage be lost but also is a valuable source of reference
material for quality control purposes.
1.3.6. Interpretation of Phage-Typing Results
The critical factors governing the interpretation of phage-typing results
are discrimination and reproducibility. In an ideal scheme the pattern of
lysis shown by isolates would be compared and any differences would

indicate that the isolates are different strains. Schemes that utilize phages
specific for a particular receptor site, such as the Vi polysaccharide of
Salmonella typhi,
are relatively reproducible and minor differences in
patterns between isolates may be indicative of distinct strains. Further-
more, highly reproducible phage lytic patterns can be assigned a
reference type number, and all isolates giving the same phage sensitivity
pattern are designated as that type (8). For example, there are 106
internationally recognized phage types of S.
typhi,
each type being
defined by a different phage pattern.
In practice, few typing schemes reach this ideal and care must be used in
interpreting the results of phage typing, All new schemes must be assessed
for their ability to discriminate between strains, i.e., to give the same pat-
tern of lysis on isolates representing the same strain and to give different
patterns on epidemiologically unrelated isolates. It is essential that the
efficacy of a new scheme is established on a well-characterized col-
lection of strains. The analysis of this data will establish how much
Bacteriophage Typing
19
flexibility should be allowed in the interpretation of lytic patterns. This
“flexibility” is introduced by applying a reaction difference rule (9). A
reaction difference is defined as a difference between a strong positive
(++) and a negative (-) reaction. The number of reaction differences that
are allowed before a pair of isolates are considered distinct is determined
by analyzing the variation in lytic patterns shown on repeated typing of
isolates that represent the same strain. It follows that the more flexibility
that is allowed, the less discriminatory power the scheme has.
The reproducibility of a phage typing scheme must be established by

repeat typing of stored isolates. Until the scheme has been shown to be
reliable, comparisons can only be made between isolates typed in the
same “run,” i.e., on the same day, on the same batch of media, and with
the same phage preparations. This chapter describes methods for the
isolation of phages from a variety of sources and techniques for quanti-
fying phage activity on appropriate host strains. In addition, we describe
protocols for the purification and propagation of phage. Last, we present
methods for the examination of lytic spectra of the bacteriophage typing
set and their use in phage typing.
2. Materials
1. Nutrient broth or solid media appropriate to the species (see Note 2). To
solidify media, add bacteriological grade agar to nutrient broth to 1.5%
(w/v); for soft agar overlays, use 0.5% (w/v). Sterilize by autoclaving at
121°C for 15 mm.
2. Double-strength nutrient broth.
3. Mitomycin C: Prepare a stock solution of 100 pg/mL in sterile water, store
in 100-200 PL aliquots in foil-wrapped bottles at -70°C. Discard the stock
after 6 mo, do not refreeze.
4. Chloroform.
5. Multiloop applicator (see Note 3).
6. Membrane filters: 0.22 pm pore size, sterile.
3. Methods
3.1. Isolation
of
Phage
3.1.1. From Sewage
1. Mix 5 mL of a 24 h composite of raw, untreated, settled sewage with 5 rnL
of double-strength nutrient broth.
2. Add 0.1 mL of broth culture of a host strain and incubate overnight at
3o”c.

20
Pitt and Gaston
3. Add 2 mL of chloroform (see Note 4), and mix vigorously for 30 s.
4. Centrifuge at 4000g for 20 min.
5. Remove the aqueous supernatant with a Pasteur pipet to a fresh contamer.
6. Filter the supernatant through a 0 22-pm membrane filter and store at 4OC
3.1.2. From Lysogenic Cultures
1. Dilute an overnight broth culture, 1 in 100, in fresh broth and incubate for
1 h at its optimal growth temperature in a water bath.
2. Add mitomycin C (see Note 5) to give a final concentration of 1 .O p,g/mL
and incubate for 4-6 h.
3. Remove the cells by centrifugation at 5OOOg.
4. Filter the culture supernatant through a 0.22~pm membrane filter and
store at 4°C.
3.2. Assay
of
Phage Activity
3.2.1. Surface Titration
1. To eight 75
x
10 mm sterile tubes add, aseptically, 0.9 mL of nutrient broth.
2. To the first tube, add 0.1 mL of phage filtrate.
3. Change the pipet tip and mix the contents gently, taking care not to
form bubbles.
4. Transfer 0.1 mL to the next tube and repeat the dilution process along
the series.
5. Using a Pasteur pipet, flood -2 mL of log phase broth culture over the
surface of a nutrient-agar plate.
6. Remove and discard the excess fluid and leave the plate open at room tem-
perature for 20-30 mm to dry.

7. Starting at tube 8, remove 20 pL of fluid and carefully dispense it onto the
surface of the agar plate (on a level surface) taking care not to form bubbles
or touch the agar.
8. Repeat the process downward to tube 1. Ensure that the drops are evenly
spaced around the plate and that they do not run over the edge of the agar
9. Replace the tip and spot 20 pL of undiluted phage filtrate on to the
seeded agar.
10. Leave the plate open at room temperature for the spots to dry and replace
the lid.
11. Incubate at 30°C overnight (see Not,: 6).
12. Score the phage/host reaction as described in Section 3.2.2., step 7.
3.2.2. Double Agar Layer
1. Prepare a series of dilutions of the phage as m Section 3.2.1.
2. Add 0.1 mL of broth culture of the host strain to 3 mL of soft agar at 45°C.
3. Workmg efficiently (do not allow the soft agar to set m the tube), rmx the
Bacteriophage Typing 21
contents of the tube by gentle mversion, and pour the bacterial suspension
over the surface of an agar plate.
4. Allow the soft agar to set and dry the surface at 37°C for 30 mm.
5. Spot 20 p,L of each phage dilution carefully onto the agar surface.
6. Incubate overnight at 30°C.
7. Examine the lawn of growth for phage activity. Score each spot using the
following classifications:
a. Confluent lysis (CL): Inhibition of all growth within dilution spot;
b. Semiconfluent lysis (SCL): Less complete with irregular edges and may
mclude some growth within the area of lysis but no individual plaques;
c. Opaque lysis (OL): Complete lysis covered by apparently resistant
bacterial overgrowth;
d. ++: more than 50 discrete plaques;
e. +: 20-50 plaques;

f. +: less than 20 plaques;
t :
-: no lysis; and
I: mhibmon of growth usually seen at low phage dilutions.
8. To determine the phage titer, record the number of countable, discrete
plaques present in a dilution spot and multiply this number by the dilution
and the volume to express the plaque forming units (pfu) per mL. For
example: 48 plaques are present on the lOA dilution (the spot from the
sixth IO-fold dilution) the titer is calculated as 48
x
lo6 x 50 (20 mL
volume = l/50 mL) = 2400
x
lo6 or 2.4 x lo9 pfu/mL. Ignore spots with
fewer than 10 plaques and count the preceding dilution. Some phage work-
ers prefer to standardize phage concentrations to routine test dtlutron (RTD,
see Note 7), but other laboratories favor the use of standard titers.
3.3. Purification of Phage
Newly
isolated phage suspensions from sewage or lysogenic strains
may contain
more than one variety of phage. This usually results in the
presence of different plaque morphologies on the host strain (see Note 8).
Individual plaques must be purified to ensure a homogeneous phage
population for subsequent propagation. Several phage strains can be
isolated from each enrichment.
1. Using a sterile toothpick or bacteriological wire, touch the center of a
well-isolated plaque and inoculate 1 mL of sterile broth in a glass tube.
Agitate the inoculator vtgorously to ensure adequate transfer of the
phage into the broth.

2. Prepare a series of lo-fold dilutions of the phage in sterile broth.
3. Spot 20-pL volumes of each dilution onto lawns of the host strain.
22 Pitt and Gaston
4. Incubate overnight at 30°C.
5. Examine the plate for purity of the plaques.
6. Repeat this process twice more and retain the last single plaque isolation
for propagation.
3.4. Propagation
of
Phage
3.4.1. Broth Culture
1. Grow the host strain in broth overnight and transfer 0.1 mL of saturated
culture to 12 mL of fresh broth.
2. Incubate the fresh culture at 37°C for 30 min, with shaking.
3. Add 0.1 mL of phage (see Note 9) to the broth culture.
4. Reincubate at 30°C with gentle or intermittent shaking for 6 h. Incubate for
longer penods, depending on the growth characteristics of the host strain.
5. Add 1 mL of chloroform (see Note 4) and shake gently.
6. Centrifuge at 4000g for 20 min and filter the supernatant.
7. Titrate the filtrate on the host strain (see Note 10).
3.4.2. Soft Agar
1. Melt 10 mL of soft nutrtent agar and cool it to 45OC.
2. Add 0.1 mL of log-phase broth culture of the host strain and 0.1 mL of
phage at RTD (see Note 7).
3. Pipet 7.5 mL of this to the surface of a large (14 cm) nutrient agar plate.
Leave for 5 min to set.
4. Incubate at 30°C overnight.
5. Flood the agar surface with 10 mL of nutrient broth and break up the soft-
agar layer with a sterile bent glass rod or Pasteur pipet.
6. Transfer the fluid to a sterile bottle and wash the agar surface with a further

10 mL of broth.
7. Pool the washings and ensure that the agar is broken up by rapid pipetmg.
8. Centrifuge the supernatant at 8OOOg and filter it.
9. Determine the phage titer.
3.5. Lytic Spectra
Phages in a typing set are best compared and characterized for host
range by the determination of their lytic spectra (see Note 11).
1. Make a 1 in 10 dilution of the phage in broth and spot 20 mL on lawns of
each of the propagating strains m the phage set.
2. After incubation at 30°C overnight, record the lytic reactions,
3. Prepare lo-fold dilutions of the phage (to titrate) and spot all the dilutions
onto each of the susceptible strains. Incubate and calculate titer.
Bacteriophage Typing
23
PHAGE
100x RTD Stook
1
Dilute to RTD
1
fill block
Em
\
Bacteria
/
Dry
30 InI”
MULTI LOOP INOCULATOR
- I ciz?
P”W UOOL
I

Incubate
30 c
0
Read reactlons
Fig. 1. Generalized scheme for phage typing of bacteria.
4. Score the reactions as follows:
a, 5: Titer equivalent to titer on homologous propagating strain;
b. 4: 10-l to 1O-2 of homologous;
c. 3: 10T3 to 10-4 of homologous;
d. 2: lo9 to lo-6 of homologous;
e. 1: Weak lyttc reaction; and
f. I: Inhibition only.
3.6. Phage Typing of Isolates
Figure 1 illustrates the stages in the phage typing of isolates.
1. Prepare 2 mL of phage stocks at 100
x
RTD and keep at 4OC (replace
every 6 mo).
2. Prepare small patches of the host strains on agar plates by “painting”
an area with a sterile swab dipped in log-phase broth culture.
24
Pitt and Gaston
3. Inoculate the center of each with 20 mL of the appropriate phage stock.
4. Incubate the plates overnight and check that each phage gives
semiconfluent lysis on its host strain.
5. Dilute each phage to RTD (prepare 4 mL of this dilution).
6. Transfer 1-2 mL of RTD phage to a sterile perspex container with wells.
7. Grow the bacterial test strains, m broth, to log phase and prepare lawns of
each on agar plates.
8. Apply phages with the aid of a multiloop applicator (see Note 3) and after

drying, incubate overnight.
9. Record phage lytic reactions for each strain.
3.7. Reaction Difference Rule
1, Select 100 isolates and type them at RTD with the phage set.
2. Record the lytic reactions and make subcultures in storage medmm.
3. Store cultures at 4°C for 1 mo, and prepare fresh broth cultures at the end
of this period.
4. Repeat the phage typing and record the reactions.
5. Prepare a histogram plot of the numbers of pairs of isolates (first and sec-
ond typing) that exhibit 0 strong differences in lytic pattern (++ vs -), 1
difference, 2 differences, and so on.
6. The reaction rule is set as the number of differences that must be allowed
so that at least 90% of the pairs of each isolate are considered “the same.”
7. Phage type sets of pairs of isolates of the same species from the same
specimen, sequenttal tsolates from the same patient, and variants (antibi-
otic, biochemical) of the same strain as tests of clinical or biological
reproducibility. Repeat the histogram to determine difference rule allowed.
4. Notes
1. Some phages are not very stable and titers may deteriorate rapidly on stor-
age. Exclude these from the phage set or check their titers more regularly.
2. For nutritionally unexacting bacteria, peptone- or tryptone-based media
are satisfactory. The nutrient quality of the medium may be increased by
the addition of beef or yeast extract, specific amino acids, or serum. Cal-
cium ions are required for optimal activity of some phages.
3. The most commonly used applicator was designed by Lidwell (10). It is avail-
able commercially from Leec Laboratories (Nottingham, UK). Phages may
be applied manually, but this is extremely labor intensive for a large number
of isolates.
4. Some phages are sensitive to chloroform, and in these cases this treatment
should be avoided. Enrichments are often more successful if chloroform is

left out. Efficient centrifugation and the availability of disposable filters
make it unnecessary to use chloroform regularly.
Bacteriophage Typing
25
5, Mitomycin C is a mutagenrc agent and gloves must be worn when handling.
The compound deteriorates in solution and is extremely light sensitive.
6. Phage activity is usually maximal at temperatures below the growth
optimum for the host species. Overgrowth of the host strain may make it
difficult to discern minute plaques and a lower incubation temperature can
help to minimize this.
7. RTD is the highest dilution of phage that just fails to give confluent lysis
of the host strain in a plate assay. This is used to standardize different
phages in a typing set to ensure similar lytic activity, because some phages
of high titer may have a low RTD and vice versa. Phage concentrations of
10, 100, or 1000 x RTD are used for some bacterial typing schemes. For
soft-agar propagation, if the RTD is 10U3 then 10 /.tL of phage stock should
be added to 10 mL of soft agar to give final concentrations equivalent to
RTD on a titration plate.
8. Heterogeneity of plaque morphology on a host strain may be due to phage
mutation and not only to different phages. Plaque morphology of pure
phages may also vary on different host strains.
9. The ratio of phage to bacterial cells is crucial for successful propagations.
A ratio of at least 2:l is advisable, but this should be determined expen-
mentally. Thus, the phage titer and the viable count of the host strain (at
the time of the addition of phage) should be carefully quantified.
10. Titers of lo8 to lOlo pfu/mL usually can be achieved, for most entero-
bacterial phages, by broth propagation; titers below this are unsatisfactory
and the propagation should be repeated by varying incubation times and
phage:bacteria ratios. Phages are easily spread in aerosols, so stocks should
be prepared with a reasonable degree of spatial or temporal separation.

11. It is important to ensure that each batch of propagations of a phage has
identical lytic propertles to the original stock. This is checked by deter-
mining its lytic spectrum on the propagating host strains of the typing set.
Batches exhibiting major discrepancies from the original must be dis-
carded, as they may contain mutated phage. Volumes (0.1 mL) of the
propagation should be lyophilized and stored in the dark for reference.
When required, reconstitute phage in 0.1 mL of broth and make up to 1 mL.
References
1. Ackermann, H W. and Dubow, M. (1987)
Viruses of Prokmyotes,
vol. 2, CRC,
Boca Raton, FL.
2 Ackermann, H. W., Auduner, A., Berthiaume, L., Jones, L.
A., Mayo, J. A.,
and
Vidaver, A. K. (1978) Guidelines for bacteriophage charactenzatlon.
Adv. Virus
Rex 23, I-24.
3. Adams, M. H
(1959) Bacteriophage,
Interscience Publishers Inc., New York.